Project Summary/Abstract: The Goal of this proposal is to dissect the mechanistic details surrounding the nucleation and early events of tau aggregation. Tau is a key misfolding and aggregating protein associated with both Alzheimer?s disease (AD) and frontotemporal dementia (FTD). There has been a significant increase in the number of people suffering from neurodegenerative diseases, including AD and FTD and is expected to continue to rise as there are currently no cures. While many studies have attributed the aggregation of tau as a cause of the disease, the process of nucleation and self-prorogation of tau aggregation is not well understood. One major challenge has been to determine the conformational basis of tau misfolding and aggregation due to the large size of the protein. To address this challenge, minimal sequence of 31 residues has been defined as the conformational nucleus responsible for the self-propagation of tau aggregation, thus providing a foundation for investigating the early stage of nucleation. With this observation we hypothesize that residues within this minimal sequence interact with other domains of the protein to induce aggregation at the single molecule level as well as interact with other misfolded tau molecules to seed aggregation. To test this, we propose to map these interactions in a site specific manner using minimally perturbing fluorescent unnatural amino acids to shed light on the early non-fibrillar oligomer formation and the local conformational dynamics over the course of aggregation. Innovation: In order to dissect site-specific interactions, kinetics and conformational dynamics surrounding the nucleation and early aggregation events of tau, new tools are needed. Unnatural amino acids sensitive to hydration are uniquely useful to probe aggregation because of the change in local hydration exhibited when once soluble protein becomes insoluble during aggregation. Unfortunately, current technologies such as fluorescent proteins and dyes are not ideal to study intramolecular events due to their prohibitive size. Unnatural amino acids chromophores offer the desired spectroscopic qualities in a framework that is non-perturbing to both structure and function of tau, making it ideal to study the intra- and intermolecular conformational dynamics, kinetics and interactions during tau aggregation. Together this will inform our direct knowledge to help elucidate the aggregation pathway.